Late stage vascular disease is characterized by inflammatory changes including the overgrowth of vascular smooth muscle cells which eventually contribute to blocking of affected arteries. Heparin is best known as an anticoagulant that has also been shown to slow smooth muscle cell growth in vitro and in selected in vivo studies. Heparin effects on vascular smooth muscle cells have been explained by several theories including heparin interactions with growth factors, heparin receptors and endocytosis of heparin to alter transcription. The PI's research group previously identified a putative heparin receptor on vascular cells and anti-receptor antibodies that mimic heparin functions in blocking DNA synthesis and decreasing ERK activation in vascular smooth muscle cells. Data from the PI's laboratory implicate this receptor and signal transduction involving cGMP production, the activation of cGMP sensitive protein kinase (PKG) and synthesis of a specific dual function phosphatase, MKP1, in heparin induced decreases in ERK activity and DNA synthesis. The proposed research will expand on preliminary results indicating Raf activity is also decreased in response to heparin treatment and will confirm that heparin works through the receptor and PKG to cause this decreased activity. Confirmation that the effects of heparin on smooth muscle cell proliferation occur through the PKG pathway will involve evaluating heparin effects on cyclin/cyclin kinase inhibitor level and cyclin D (G1 phase cyclin) synthesis using cGMP mimics and PKG inhibitors. Additional results from the PI's laboratory demonstrate cytoskeletal localization of a subset of active JNK and p38 stress activated protein kinase enzymes and the ability of heparin to decrease activation of JNK and p38 as assessed by western blotting. Proposed research will determine whether heparin also results in decreased activity of the cytoskeletal associated JNK and p38 and effectors found in the cytoplasm, or whether heparin effects are restricted to the nucleus where the MKP1 resides. To further evaluate the extent to which heparin treatment activates cGMP signaling through PKG, known PKG targets such as VASP and PAK1 will be evaluated to determine whether they are specifically phosphorylated in response to heparin treatment as they are after increases in NO or artificial activators of the PKG enzyme and the possibility of a shared set of changes in gene expression will be evaluated. Preliminary results obtained in the PI's laboratory indicate that PKG levels decrease after heparin treatment suggesting that PKG degradation might be induced by heparin signaling and PKG activation. The possibility that this occurs will involve use of proteasome inhibitors and methods to detect ubiquitin linked to PKG. Finally, this study will continue work aimed at isolating the heparin receptor and obtaining sequence information that will facilitate gene identification. Together, the proposed studies will confirm the importance of the heparin receptor in heparin induced changes in vascular smooth muscle cells and will provide a significant advancement in understanding of how heparin alters sensitive vascular smooth muscle cells. One of the most complicated informational systems in blood vessels is that which controls cell growth and division that can lead both to damage repair, and when over active, to vascular disease. The proposed research continues a pattern of investigation in the PI's laboratory focused on understanding the way in which the information transfer leading to cell growth is normally turned off with a focus on the anti-coagulant molecule heparin. A better understanding of these negative pathways at the molecular level, including the pathway turned on by heparin and following through protein kinase G already known for its involvement in reducing blood vessel tension, will be helpful in designing improved treatments for vascular disease. [unreadable] [unreadable] [unreadable] [unreadable]